Therapeutic cancer vaccination is a type of immunotherapy. Immunotherapy is a new treatment modality that is emerging to join chemotherapy, radiation and surgery as a class of drugs for treatment of cancer. Immunotherapy methods seek to harness the power of the immune system in order to treat diseases. Therapeutic vaccination is a type of immunotherapy that is potentially curative therapy against existing tumors, viruses and bacterial pathogens. Therapeutic vaccines generally consist of an antigen source derived from the target disease and an adjuvant designed to enhance the desired immune response. In some therapeutic vaccine protocols, living immune cells, either derived from the host (autologous) or from a donor (allogeneic), are components of therapeutic vaccines. Autologous and allogeneic living immune cells, such as dendritic cells, NK cells and T-cells, are also used in immunotherapy protocols for the treatment of cancer.
Lymphocytes are part of the vertebrate immune system and include large granular lymphocytes and small lymphocytes. Large granular lymphocytes include natural killer cells (NK cells). Small lymphocytes consist of T-cells and B cells. NK cells are part of the innate immune system and play a major role in defending an animal from tumors and virally infected cells. NK cells can distinguish infected and tumor cells from uninfected cells through the multihistocompatibility complex (MHC) class I surface molecules. NK cells are activated in response to cytokines and upon activation release cytotoxic granules such as perforin and granzyme B that destroy the infected cells or tumor cells. NK cells are characterized by the cell surface markers CD16+ and CD56+, but do not express CD4.
T-cells and B-cells are part of the adaptive immune response and recognize non-self antigens. B-cells respond to non-self antigen by antibody production. Different types of T-cells respond to the non-self antigens in different ways. Cytotoxic T-cells (CTL) are characterized by the markers CD3+, CD8+ and TCRαβ, and do not express CD4. Tumor-specific CD8+ CTLs are mainly responsible for tumor elimination. Moreover, CTLs can specifically recognize tumor cells and do not attack normal cells of the same tissue. CTLs produce toxic granules that contain powerful enzymes including granzyme B and perforin that induce death of diseased or cancerous cells.
T helper cells are characterized by the cell surface markers CD3+, CD4+ and TCRαβ. T-helper cells produce cytokines or other molecules that direct the immune response through other cells or molecules. T-helper cells are not known to directly mediate killing of cells and thus do not normally contain cytoxic granules such as granzyme B or perforin. T-helper cells are sub-categorized into two types: T-helper type 1 (Th1) and the T-helper type 2 (Th2). Th1 cells mediate cellular immunity and are critical for immune-mediated tumor eradication, whereas Th2 cells mediate a humoral or antibody immunity. Th1 and Th2 cells are counter-regulatory, increased Th1 cells down regulate Th2 cells and vice versa.
A variety of immunotherapy methods and compositions have been developed in order to enhance or suppress the immune response in patients. Cell therapy methods often involve ex-vivo manipulations such as proliferation, differentiation and/or activation of cells and the transfer of these cells to a patient as therapy.
Adoptive immunotherapy involves removing lymphocytes from the patient, boosting their anti-cancer activity ex-vivo, growing the cells to large clinically relevant numbers, and then returning the cells to the patient:
Initial experiments in adoptive immunotherapy involved removing lymphocytes from the blood of a patient and growing them in the presence of the lymphokine interleukin-2 (IL-2), an immune stimulator. The cells were then returned to the patient. These lymphocytes were called lymphokine-activated killer (LAK) cells.
A stronger response against tumor cells was obtained using lymphocytes isolated from the tumor itself. These tumor-infiltrating lymphocytes (TILs) are grown in the presence of IL-2 and returned to the body to attack the tumor.
Adoptive immunotherapy is a form of immunotherapy where ex vivo processed cells are introduced into the body. Pre-clinical studies suggested anti-tumor activity could be enhanced by using interleukin-2 together with ex vivo activated and expanded autologous lymphocytes. Also, the first objective responses with high-dose bolus interleukin-2 therapy were noted in patients receiving interleukin-2 together with LAK cells prepared through in vitro activation of autologous peripheral blood lymphocytes that were harvested by lymphopheresis, and initially, it appeared that the combination of interleukin-2/LAK was more active than interleukin-2 alone. IL-2 is a cytokine produced by Th1 helper cells. Interest in the use of adoptive immunotherapy declined after clinical studies failed to demonstrate that the addition of activated LAK or TIL cells with IL-2 is any more effective than IL-2 alone.
CD4+T (Th) cells are crucial for the activation and regulation of most aspects of the host defense against infections and for adequate function of cytotoxic CD8+ lymphocytes (CTL). Much of the research in tumor immunology has been focused on naturally occurring autologous tumor-specific T cells that are predominantly CD8+ and can be isolated from melanoma and some other tumors. These cells can be expanded in vitro and reinfused. This type of adoptive immunotherapy has resulted in objective tumor responses and long-term survival in some patients whose disease is refractory to other interventions. While there is an extensive clinical experience using CD8+ cytotoxic T cells as adoptive immunotherapy, poor clinical responses underscore the need to improve these therapies in order to achieve tumor rejection in higher percentages of patients.
CD8+ lymphocytes can fail to maintain functionality in vivo in large part because of the absence of CD4+ T cell help. In vitro, CD8+ cells release large quantities of interferon-γ (IFN-γ) upon exposure to MHC-compatible cell lines and lyse autologous antigen-positive and MHC class I-positive tumors. However, the genetic instability of tumor cells frequently leads to losses in the ability to process and present endogenous antigens rendering tumors inherently unreliable targets for CD8+ cytolytic T cells. Furthermore, CD8+ T cells appear to lack the intrinsic ability to orchestrate a broad antitumor response that seems inherent in some CD4+ T cell subsets.
While immunotherapies with adoptive transfer of CTL have demonstrated promise in many animal models, the translation of these results to humans has proven to be difficult and elusive. CD4+ cells, especially of the Th1 subtype, would be an attractive addition to the available immunotherapy armamentarium to provide natural “help” to adoptively transferred CD8+ killer cells. However, there are significant barriers in working with naturally occurring tumor-specific CD4+ cells. The genetic diversity of the class II HLA associated with CD4+ helper cells in any given population of patients is much more complex than in the case of class I HLA found in CD8+ cytolytic (killer) T-cells, making identification of epitopes and TCRs more problematic. Moreover, CD4+ T-cells expand in vitro less well than CD8+ cells and the culture conditions significantly affect their characteristics. Finally, there is a paucity of realistic animal models based on tumor-specific CD4+ cells. These factors have limited translational research using CD4+ T cells and limited their clinical use.
This invention discloses a method for producing clinically-relevant numbers of CD4+ cells that also contain the cytolytic granules granzyme B and perforin and function both as Th1 cells (produce IFN-gamma and not IL-4) and have NK-like activity (recognize and kill tumor cells and not normal cells) for use in therapeutic cancer vaccine and adoptive immune cell therapy protocols.